Lipase Variants and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to isolated lipase variants, comprising a substitution at one or more positions corresponding to positions I86D,E,N,Q, E87C, I90D,E,Q, and N92D,E,Q of the mature polypeptide of SEQ ID NO: 2, wherein the variant has lipase activity. In some embodiments the present invention relates to isolated polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; methods of producing the variants; and compositions comprising the variants. The present invention also relates to methods of obtaining lipase variants; methods of cleaning; and use of lipase variants for cleaning.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to lipase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants.

2. Description of the Related Art

Lipases are included in detergent compositions to increase washperformance and specifically to improve lipid stain removal. Buildersare also included in detergent compositions amongst other for thepurpose of lowering the concentration of calcium which due toprecipitation may lead to “graying” of the treated surfaces. Low levelsof calcium have shown to result in a reduction of lipase activity.

The catalytic site in many lipases is shielded by a lid domain (lidregion or lid) and studies have indicated that the lid is important forlipase activity and has a role in activation of lipases. Enhancedcatalytic activity in the presence of a water/lipid interface isreferred to as “interfacial activation” and describes the situationwhere the amphiphilic surface loop i.e. the lid opens on contact withthe interface. Shu et al. Enzyme and Microbial Technology 48 (2011)129-133 generated four Aspergillus niger lipase (ANL) mutants one withno lid and three with the lid in an open conformation for the purpose ofidentifying interfacial activation independent lipase mutants.

There is thus a need for lipases with improved lipase activity inparticular for use in detergent compositions.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that mutations in the lid regionof a parent lipase leads to lipase variants as described herein with analtered activation, more specifically the variants have in someembodiments of the invention an improved specific activity at lowconcentrations of calcium as compared to the parent lipase and in someembodiments of the invention the variants have an improved washperformance as compared to the parent lipase.

The present invention relates to isolated lipase variants, comprising asubstitution at one or more positions corresponding to positions 86, 87,90, and/or 92, especially substitutions to D, E, Q, N, or C, andspecifically substitutions 186D,E,N,Q, E87C, 190D,E,Q, and N92D,E,Q ofthe mature polypeptide of SEQ ID NO: 2, wherein the variant has lipaseactivity.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; methods of producing the variants; andcompositions comprising the variants.

The present invention also relates to methods of obtaining lipasevariants; methods of cleaning; and use of lipase variants for cleaning.

DEFINITIONS

Lipase: The term “lipase” or “lipolytic enzyme” or “lipid esterase” isan enzyme in class EC3.1,1 as defined by Enzyme Nomenclature. It mayhave lipase activity (triacylglycerol lipase, EC3.1.1.3), cutinaseactivity (EC3.1.1.74), sterol esterase activity (EC3.1.1.13) and/orwax-ester hydrolase activity (EC3.1.1.50). For purposes of the presentinvention, lipase activity is determined according to the proceduredescribed in the Examples. In one aspect, the variants of the presentinvention have at least 20%, e.g., at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 100% of the lipase activityof the mature polypeptide of SEQ ID NO: 2 or 3.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has lipaseactivity. In one aspect, a fragment contains at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, and at least 95% of the number of amino acidsof the mature polypeptide.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to: improved catalyticrate; improved specific activity; improved substrate cleavage; improvedopening of the lid; reduced calcium ion dependency; improved buildertolerance, and/or improved wash performance.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 269 of SEQ ID NO: 2. In one aspect, themature polypeptide is amino acids 1 to 269 of SEQ ID NO: 3.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lipase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 67 to 873 of SEQ ID NO: 1.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent lipase: The term “parent” or “parent lipase” means alipase to which an alteration is made to produce the enzyme variants ofthe present invention. The parent may be a naturally occurring(wild-type) polypeptide or a variant or fragment thereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”. For purposes of the present invention, the sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having lipase activity. In one aspect, a subsequence containsat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, and at least 95% ofthe number of nucleotides of the mature polypeptide coding sequence.

Variant: The term “variant” means a polypeptide having lipase activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding one or more (e.g., several)amino acids adjacent to and immediately following the amino acidoccupying a position. The variants of the present invention have atleast 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or at least 100% of the lipaseactivity of the mature polypeptide of SEQ ID NO: 2 or 3.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type lipase: The term “wild-type” lipase means a lipase expressedby a naturally occurring microorganism, such as a bacterium, yeast, orfilamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another lipase. The amino acid sequence of another lipase isaligned with the mature polypeptide disclosed in SEQ ID NO: 2, and basedon the alignment, the amino acid position number corresponding to anyamino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2is determined using the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleprogram of the EMBOSS package (EMBOSS: The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),preferably version 5.0.0 or later. The parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSSversion of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in another lipasecan be determined by an alignment of multiple polypeptide sequencesusing several computer programs including, but not limited to, MUSCLE(multiple sequence comparison by log-expectation; version 3.5 or later;Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066;Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh,2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods inMolecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later; Thompsonet al., 1994, Nucleic Acids Research 22: 4673-4680), using theirrespective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402).

Even greater sensitivity can be achieved if the family or superfamilyfor the polypeptide has one or more representatives in the proteinstructure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol.Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from a variety of sources (PSI-BLAST,secondary structure prediction, structural alignment profiles, andsolvation potentials) as input to a neural network that predicts thestructural fold for a query sequence. Similarly, the method of Gough etal., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequenceof unknown structure with the superfamily models present in the SCOPdatabase. These alignments can in turn be used to generate homologymodels for the polypeptide, and such models can be assessed for accuracyusing a variety of tools developed for that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411 Phe” or “G205R+S411F”, representingsubstitutions at positions 205 and 411 of glycine (G) with arginine (R)and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of arginine and glycine at positions 170 and 195 withtyrosine and glutamic acid, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly, Ala+Arg170Gly,Ala” designates thefollowing variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”,“Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated lipase variants, comprising asubstitution at one or more (e.g., several) positions corresponding topositions 186D,E,N,Q; E87C; 190D,E,Q; and N92D,E,Q of the maturepolypeptide of SEQ ID NO: 2, wherein the variant has lipase activity.

Most lipases become fully activated upon binding to a water/lipidinterface and remain virtually inactive when the substrate is in asoluble, mono-phasic state. This phenomenon is referred to as“interfacial activation”. Interfacial activation is the phenomenon bywhich the activity of the lipase increases dramatically when thesubstrate reaches concentrations beyond its critical micelleconcentration (CMC). At this state the substrate molecules aggregate andform micelles on which the enzyme can bind. The catalytic triad isshielded from the external environment by a helical secondary structure,the 3^(rd) alfa helix in the protein, called the “lid”. Studies haveindicated that this lid domain, herein defined as the positionscorresponding to amino acid 82-98 of the mature polypeptide of SEQ IDNO: 2 (amino acid 81-97 in SEQ ID NO: 3), plays a central role in theinactive/active states of the enzyme. The lid opens upon interfacialactivation enabling access of the substrate to the catalytic triad. Asthe lid opens a large hydrophobic surface area representing ˜10% of thetotal surface area of the enzyme is exposed, which are thought toposition the enzyme optimally for catalysis. Activity measurements,X-ray crystal structures and MD simulations on Thermomyces lanoginosuslipase and related lipases suggest that the opening of the lid isenergetically unfavorable in aqueous solutions.

Variants

The present invention (also) provides lipase variants, comprising asubstitution at one or more (e.g., several) positions corresponding topositions 186D,E,N,Q; E87C; 190D,E,Q; and N92D,E,Q, wherein the varianthas lipase activity.

In an embodiment, the variant has sequence identity of at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, to the amino acid sequence of the parentlipase.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the mature polypeptide of SEQ ID NO: 2or 3.

In one aspect, the number of alterations in the variants of the presentinvention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 alterations.

In another aspect, a variant comprises a substitution at one or more(e.g., several) positions corresponding to positions 186D,E,N,Q; E87C;190D,E,Q; and N92D,E,Q. In another aspect, a variant comprises asubstitution at two positions corresponding to any of positions186D,E,N,Q; E87C; 190D,E,Q; and N92D,E,Q. In another aspect, a variantcomprises a substitution at three positions corresponding to any ofpositions 186D,E,N,Q; E87C; 190D,E,Q; and N92D,E,Q. In another aspect, avariant comprises a substitution at each position corresponding topositions 186D,E,N,Q; E87C; 190D,E,Q; and N92D,E,Q.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 86 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the amino acid at a positioncorresponding to position 86 is substituted with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val, preferably with Asn, Asp, Gln, or Glu. In another aspect,the variant comprises or consists of the substitution 186D, 186E, 186N,or 186Q of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 87 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the amino acid at a positioncorresponding to position 87 is substituted with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val, preferably with Cys. In another aspect, the variantcomprises or consists of the substitution E87C of the mature polypeptideof SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 90 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the amino acid at a positioncorresponding to position 90 is substituted with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val, preferably with Asp, Gln, or Glu. In another aspect, thevariant comprises or consists of the substitution at a positioncorresponding to position 190D, 190E, or 190Q of the mature polypeptideof SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 92 of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the amino acid at a positioncorresponding to position 92 is substituted with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val, preferably with Asp, Gln, or Glu. In another aspect, thevariant comprises or consists of the substitution at a positioncorresponding to position N92D, N92E, or N92Q of the mature polypeptideof SEQ ID NO: 2.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 87, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 90, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 92, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 87 and 90, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 87 and 92, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 90 and 92, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86, 87, and 90, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86, 87, and 92, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86, 90 and 92, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 87, 90, and 92, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86, 87, 90, and 92, such as thosedescribed above.

In another aspect, the variant comprises or consists of one or more(e.g., several) substitutions selected from the group consisting ofpositions corresponding to position 186D,E,N,Q; E87C; 190D,E,Q; andN92D,E,Q of the mature polypeptide of SEQ ID No: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions 186D,E,N,Q+E87C ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions186D,E,N,Q+190D,E,Q of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions186D,E,N,Q+N92D,E,Q of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions E87C+190D,E,Q ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions E87C+N92D,E,Q ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions 190D,E,Q+N92D,E,Qof the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions186D,E,N,Q+E87C+190D,E,Q of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions186D,E,N,Q+E87C+N92D,E,Q of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions186D,E,N,Q+190D,E,Q+N92D,E,Q of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positionsE87C+190D,E,Q+N92D,E,Q of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of thesubstitutions at positions corresponding to positions186D,E,N,Q+E87C+190D,E,Q+N92D,E,Q of the mature polypeptide of SEQ IDNO: 2.

The variants may further comprise one or more additional substitutionsat one or more (e.g., several) other positions. In one aspect theinvention relates to variants, which comprises one or more substitutionsselected from the group consisting of D62C; S83T; R84G; 186P; E87T,A,Q;W89L; G91L,A; L93T; N94D,S; F95Y; D96T,I; L97P,F; K98Q,D,T correspondingto the mature polypeptide of SEQ ID NO: 2

In one aspect the invention relates to variants, which consists orcomprises the set of substitutions corresponding to positions of themature polypeptide of SEQ ID NO: 2 as listed below:

TABLE 1 S83T + R84G + I86D + E87T + W89L + I90Q + G91L + N92D + L93T +F95Y + D96T + K98T; S83T + E87T + G91L + N92D + F95Y + D96T + L97P +K98Q; I86P + E87A + G91A + N94D + D96I + L97F + K98D; S83T + R84G +I86D + E87Q + W89L + I90Q + G91L + N92E + L93T + N94S + F95Y + D96T +K98T; D62C + S83T + R84G + I86D + E87C + W89L + I90Q + G91L + N92D +L93T + F95Y + D96T + K98T; D62C + S83T + E87C + G91L + N92D + F95Y +D96T + L97P + K98Q; D62C + I86P + E87C + G91A + N94D + D96I + L97F +K98D; D62C + S83T + R84G + I86D + E87C + W89L + I90Q + G91L + N92E +L93T + N94S + F95Y + D96T + K98T. S83T + R84G + I86D + E87T + W89L +I90Q + G91L + N92D + L93T + F95Y + D96T + K98T + T231R + N233R; S83T +E87T + G91L + N92D + F95Y + D96T + L97P + K98Q + T231R + N233R; I86P +E87A + G91A + N94D + D96I + L97F + K98D + T231R + N233R; S83T + R84G +I86D + E87Q + W89L + I90Q + G91L + N92E + L93T + N94S + F95Y + D96T +K98T + T231R + N233R; D62C + S83T + R84G + I86D + E87C + W89L + I90Q +G91L + N92D + L93T + F95Y + D96T + K98T + T231R + N233R; D62C + S83T +E87C + G91L + N92D + F95Y + D96T + L97P + K98Q + T231R + N233R; D62C +I86P + E87C + G91A + N94D + D96I + L97F + K98D + T231R + N233R; orD62C + S83T + R84G + I86D + E87C + W89L + I90Q + G91L + N92E + L93T +N94S + F95Y + D96T + K98T + T231R + N233R.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, e.g., by H. Neurath and R. L. Hill,1979, In, The Proteins, Academic Press, New York. Common substitutionsare Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val,Ala/Glu, and Asp/Gly.

Alternatively, the amino acid substitutions are of such a nature thatthe physico-chemical properties of the polypeptides are altered orimproved. For example, amino acid changes may alter or improve thecatalytic rate, specific activity, and substrate cleavage.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lipase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, e.g., de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identity of essential amino acids can also beinferred from an alignment with a related polypeptide.

In an embodiment, the variant has improved catalytic rate compared tothe parent enzyme.

In an embodiment, the variant has improved specific activity compared tothe parent enzyme.

In an embodiment, the variant has improved substrate cleavage comparedto the parent enzyme.

In an embodiment, the variant has improved opening of the lid comparedto the parent enzyme.

In an embodiment, the variant has reduced calcium ion dependency, i.e. ahigher calcium ion independency as compared to the parent enzyme.

In an embodiment, the variant has improved builder tolerance as comparedto the parent enzyme. The builder may be selected from the group ofbuilders mentioned in the section “Builders” below. In an embodiment,the variant has improved EDTA tolerance as compared to the parentenzyme. In an embodiment, the variant has improved chelating agentand/or crystal growth inhibitor tolerance as compared to the parentenzyme. The chelating agent and/or crystal growth inhibitor may beselected from the group of chelating agent and crystal growth inhibitormentioned in the section “Chelating agents and Crystal growthinhibitors” below.

In an embodiment, the variant has improved wash performance compared tothe parent enzyme.

Parent Lipases

The parent lipase may be (a) a polypeptide having at least 60% sequenceidentity to the mature polypeptide of SEQ ID NO: 2 or 3; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, or (ii) the full-length complement of (i); or (c) apolypeptide encoded by a polynucleotide having at least 60% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 or 3 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which havelipase activity. In one aspect, the amino acid sequence of the parentdiffers by no more than 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 from the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2 or 3. In another aspect, the parent comprisesor consists of the mature polypeptide of SEQ ID NO: 2 or 3. In anotheraspect, the parent comprises or consists of amino acids 1 to 269 of SEQID NO: 2. In another aspect, the parent comprises or consists of aminoacids 1 to 269 of SEQ ID NO: 3.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 2 or 3 containing at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, and at least 95% of the number of amino acids of the maturepolypeptide.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2 or 3.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or 3, or a fragment thereof, may be usedto design nucleic acid probes to identify and clone DNA encoding aparent from strains of different genera or species according to methodswell known in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(e.g., with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, e.g., X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe is67 to 873 of SEQ ID NO: 1. In another aspect, the nucleic acid probe isa polynucleotide that encodes the polypeptide of SEQ ID NO: 2 or 3; themature polypeptide thereof; or a fragment thereof. In another aspect,the nucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial lipase. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces lipase, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma lipase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis lipase.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus lipase.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans lipase.

The parent may be a fungal lipase. For example, the parent may be ayeast lipase such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia lipase; or a filamentous fungal lipasesuch as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria lipase.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis lipase.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa(Thermomyces lanuginosus), Irpex lacteus, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium funiculosum, Penicilliumpurpurogenum, Phanerochaete chrysosporium, Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia setosa, Thielavia spededonium, Thielaviasubthermophila, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma viride lipase.

In another aspect, the parent is a Thermomyces lanuginosus lipase, e.g.,the lipase of SEQ ID NO: 2 or the mature polypeptide thereof. In anotheraspect, the parent is a Rhizomucor miehei lipase, e.g., the lipase ofSEQ ID NO: 3 or the mature polypeptide thereof.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving lipase activity, comprising: (a) introducing into a parent lipasea substitution at one or more (e.g., several) positions corresponding topositions 186D,E,N,Q, E87C, 190D,E,Q, and N92D,E,Q of the maturepolypeptide of SEQ ID NO: 2, wherein the variant has lipase activity;and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO95/17413; or WO95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO96/00787), Fusariumvenenatum amyloglucosidase (WO00/56900), Fusarium venenatum Daria(WO00/56900), Fusarium venenatum Quinn (WO00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO94/25612) and a Bacillus subtilisSP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular. Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis daI genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980). The yeasthost cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative. The filamentous fungal host cellmay be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium Mops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. The variant may be purified by a variety of proceduresknown in the art including, but not limited to, chromatography (e.g.,ion exchange, affinity, hydrophobic, chromatofocusing, and sizeexclusion), electrophoretic procedures (e.g., preparative isoelectricfocusing), differential solubility (e.g., ammonium sulfateprecipitation), SDS-PAGE, or extraction (see, e.g., ProteinPurification, Janson and Ryden, editors, VCH Publishers, New York, 1989)to obtain substantially pure variants. In an alternative aspect, thevariant is not recovered, but rather a host cell of the presentinvention expressing the variant is used as a source of the variant.

Compositions

The compositions of the invention are in particular solid or liquidcleaning and/or treatment compositions. In a preferred aspect of theinvention, the composition has a single or multi-compartment unit doseform.

The non-limiting list of composition components illustrated hereinafterare suitable for use in the compositions and methods herein may bedesirably incorporated in certain embodiments of the invention, e.g. toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the composition as is thecase with perfumes, colorants, dyes or the like. The levels of any suchcomponents incorporated in any fabric and home care compositions are inaddition to any materials previously recited for incorporation. Theprecise nature of these additional components, and levels ofincorporation thereof, will depend on the physical form of thecomposition and the nature of the cleaning operation for which it is tobe used. Suitable component materials include, but are not limited to,surfactants, builders, chelating agents, dye transfer inhibiting agents,dispersants, enzymes, and enzyme stabilizers, catalytic materials,bleach activators, hydrogen peroxide, sources of hydrogen peroxide,preformed peracids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,hueing dyes, perfumes, perfume delivery systems, structure elasticizingagents, fabric softeners, carriers, hydrotropes, processing aids,solvents and/or pigments. In addition to the disclosure below, suitableexamples of such other components and levels of use are found in U.S.Pat. No. 5,576,282; U.S. Pat. No. 6,306,812; and U.S. Pat. No. 6,326,348that are incorporated by reference.

Thus, in certain embodiments the invention do not contain one or more ofthe following adjuncts materials: surfactants, builders, chelatingagents, dye transfer inhibiting agents, dispersants, additional enzymes,and enzyme stabilizers, catalytic materials, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, preformed peracids, polymericdispersing agents, clay soil removal/anti-redeposition agents,brighteners, suds suppressors, dyes, perfumes, perfume delivery systems,structure elasticizing agents, fabric softeners, carriers, hydrotropes,processing aids, solvents and/or pigments. However, when one or morecomponents are present, such one or more components may be present asdetailed below:

Builders—

The compositions of the present invention may comprise one or moredetergent builders, co-builders, builder systems or a mixture thereof.When a builder is used, the detergent composition will typicallycomprise at least 1%, from 2% to 60% or from 5% to 10% builder byweight. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. The composition may be substantially freeof builder; substantially free means “no deliberately added” zeoliteand/or phosphate. Typical zeolite builders include zeolite A, zeolite Pand zeolite MAP. A typical phosphate builder is sodiumtri-polyphosphate.

The builder and/or co-builder may particularly be a chelating agent thatforms water-soluble complexes with Ca and Mg. Any builder and/orco-builder known in the art for use in detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The detergent composition may include include a co-builder alone, or incombination with a builder, e.g. a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid(SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamicacid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diaceticacid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diaceticacid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilicacid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA),taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid(SMDA), N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA),diethanolglycine (DEG), Diethylenetriamine Penta (Methylene Phosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO09/102,854, U.S. Pat. No.5,977,053

Chelating Agents and Crystal Growth Inhibitors—

The compositions herein may contain a chelating agent and/or a crystalgrowth inhibitor. Suitable molecules include copper, iron and/ormanganese chelating agents and mixtures thereof. Suitable moleculesinclude DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethanediphosphonic acid), DTPMP (Diethylene triamine penta(methylenephosphonic acid)), 1,2-Dihydroxybenzene-3,5-disulfonic acid disodiumsalt hydrate, ethylenediamine, diethylene triamine,ethylenediaminedisuccinic acid (EDDS),N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin and2-Phosphonobutane 1,2,4-tricarboxylic acid (Bayhibit® AM) andderivatives thereof. Typically the composition may comprise from 0.005%to 15% or from 3.0% to 10% chelating agent or crystal growth inhibitorby weight of the composition.

Surfactants—

The compositions according to the present invention may comprise asurfactant or surfactant system wherein the surfactant can be selectedfrom nonionic surfactants, anionic surfactants, cationic surfactants,ampholytic surfactants, zwitterionic surfactants, semi-polar nonionicsurfactants and mixtures thereof. When present, surfactant is typicallypresent at a level of from 0.1% to 60%, from 1% to 50% or from 5% to 40%by weight of the composition.

Suitable anionic detersive surfactants include sulphate and sulphonatedetersive surfactants.

Suitable sulphonate detersive surfactants include alkyl benzenesulphonate, in one aspect, C₁₀₋₁₃ alkyl benzene sulphonate. Suitablealkyl benzene sulphonate (LAS) may be obtained, by sulphonatingcommercially available linear alkyl benzene (LAB); suitable LAB includeslow 2-phenyl LAB, such as those supplied by Sasol under the tradenameIsochem® or those supplied by Petresa under the tradename Petrelab®,other suitable LAB include high 2-phenyl LAB, such as those supplied bySasol under the tradename Hyblene®. A suitable anionic detersivesurfactant is alkyl benzene sulphonate that is obtained by DETALcatalyzed process, although other synthesis routes, such as HF, may alsobe suitable. In one aspect a magnesium salt of LAS is used.

Suitable sulphate detersive surfactants include alkyl sulphate, in oneaspect, C₈₋₁₈ alkyl sulphate, or predominantly C₁₂ alkyl sulphate.

Another suitable sulphate detersive surfactant is alkyl alkoxylatedsulphate, in one aspect, alkyl ethoxylated sulphate, in one aspect, aC₈₋₁₈ alkyl alkoxylated sulphate, in another aspect, a C₈₋₁₈ alkylethoxylated sulphate, typically the alkyl alkoxylated sulphate has anaverage degree of alkoxylation of from 0.5 to 20, or from 0.5 to 10,typically the alkyl alkoxylated sulphate is a C₈₋₁₈ alkyl ethoxylatedsulphate having an average degree of ethoxylation of from 0.5 to 10,from 0.5 to 7, from 0.5 to 5 or even from 0.5 to 3.

The alkyl sulphate, alkyl alkoxylated sulphate and alkyl benzenesulphonates may be linear or branched, substituted or un-substituted.

The detersive surfactant may be a mid-chain branched detersivesurfactant, in one aspect, a mid-chain branched anionic detersivesurfactant, in one aspect, a mid-chain branched alkyl sulphate and/or amid-chain branched alkyl benzene sulphonate, e.g. a mid-chain branchedalkyl sulphate. In one aspect, the mid-chain branches are C₁₋₄ alkylgroups, typically methyl and/or ethyl groups.

Suitable non-ionic detersive surfactants are selected from the groupconsisting of: C₈-C₁₈ alkyl ethoxylates, such as, NEODOL® non-ionicsurfactants from Shell; C₆-C₁₂ alkyl phenol alkoxylates wherein thealkoxylate units may be ethyleneoxy units, propyleneoxy units or amixture thereof; C₁₂-C₁₈ alcohol and C₆-C₁₂ alkyl phenol condensateswith ethylene oxide/propylene oxide block polymers such as Pluronic®from BASF; C₁₄-C₂₂ mid-chain branched alcohols; C₁₄-C₂₂ mid-chainbranched alkyl alkoxylates, typically having an average degree ofalkoxylation of from 1 to 30; alkylpolysaccharides, in one aspect,alkylpolyglycosides; polyhydroxy fatty acid amides; ether cappedpoly(oxyalkylated) alcohol surfactants; and mixtures thereof.

Suitable non-ionic detersive surfactants include alkyl polyglucosideand/or an alkyl alkoxylated alcohol.

In one aspect, non-ionic detersive surfactants include alkyl alkoxylatedalcohols, in one aspect C₈₋₁₈ alkyl alkoxylated alcohol, e.g. a C₈₋₁₈alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol may have anaverage degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may bea C₈₋₁₈ alkyl ethoxylated alcohol having an average degree ofethoxylation of from 1 to 10, from 1 to 7, more from 1 to 5 or from 3 to7. The alkyl alkoxylated alcohol can be linear or branched, andsubstituted or un-substituted. Suitable nonionic surfactants includethose sold under the tradename Lutensol® from BASF.

Suitable cationic detersive surfactants include alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof.

Suitable cationic detersive surfactants are quaternary ammoniumcompounds having the general formula: (R)(R₁)(R₂)(R₃)N⁺X⁻, wherein, R isa linear or branched, substituted or unsubstituted C₆₋₁₈ alkyl oralkenyl moiety, R₁ and R₂ are independently selected from methyl orethyl moieties, R₃ is a hydroxyl, hydroxymethyl or a hydroxyethylmoiety, X is an anion which provides charge neutrality, suitable anionsinclude: halides, e.g. chloride; sulphate; and sulphonate. Suitablecationic detersive surfactants are mono-C₆₋₁₈ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chlorides. Highly suitable cationicdetersive surfactants are mono-C₈₋₁₀ alkyl mono-hydroxyethyl di-methylquaternary ammonium chloride, mono-C₁₀₋₁₂ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chloride and mono-C₁₀ alkylmono-hydroxyethyl di-methyl quaternary ammonium chloride.

Suitable amphoteric/zwitterionic surfactants include amine oxides andbetaines. Amine-neutralized anionic surfactants—Anionic surfactants ofthe present invention and adjunct anionic cosurfactants, may exist in anacid form, and said acid form may be neutralized to form a surfactantsalt which is desirable for use in the present detergent compositions.Typical agents for neutralization include the metal counterion base suchas hydroxides, eg, NaOH or KOH. Further preferred agents forneutralizing anionic surfactants of the present invention and adjunctanionic surfactants or cosurfactants in their acid forms includeammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitablenon-limiting examples including monoethanolamine, diethanolamine,triethanolamine, and other linear or branched alkanolamines known in theart; e.g., highly preferred alkanolamines include 2-amino-1-propanol,1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amineneutralization may be done to a full or partial extent, e.g. part of theanionic surfactant mix may be neutralized with sodium or potassium andpart of the anionic surfactant mix may be neutralized with amines oralkanolamines.

Surfactant systems comprising mixtures of one or more anionic and inaddition one or more nonionic surfactants optionally with an additionalsurfactant such as a cationic surfactant, may be preferred. Preferredweight ratios of anionic to nonionic surfactant are at least 2:1, or atleast 1:1 to 1:10.

Bleach Component—

The bleach component suitable for incorporation in the methods andcompositions of the invention comprise one or a mixture of more than onebleach components. Suitable bleach components include bleachingcatalysts, photobleaches, bleach activators, hydrogen peroxide, sourcesof hydrogen peroxide, pre-formed peracids and mixtures thereof. Ingeneral, when a bleach component is used, the compositions of thepresent invention may comprise from 0.00001 to 90 wt %, 0.0001% to 50%or from 0.001% to 25% bleach component by weight of the subject cleaningcomposition. Examples of suitable bleach components include:

(1) Pre-formed peracids: Suitable preformed peracids include, but arenot limited to, compounds selected from the group consisting ofpre-formed peroxyacids or salts thereof, typically either aperoxycarboxylic acid or salt thereof, or a peroxysulphonic acid or saltthereof.

The pre-formed peroxyacid or salt thereof is preferably aperoxycarboxylic acid or salt thereof, typically having a chemicalstructure corresponding to the following chemical formula:

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁴ group can be linear or branched,substituted or unsubstituted; and Y is any suitable counter-ion thatachieves electric charge neutrality, preferably Y is selected fromhydrogen, sodium or potassium. Preferably, R¹⁴ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably, the peroxyacid orsalt thereof is selected from peroxyhexanoic acid, peroxyheptanoic acid,peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any saltthereof, or any combination thereof. Particularly preferred peroxyacidsare phthalimido-peroxy-alkanoic acids, in particular ε-phthahlimidoperoxy hexanoic acid (PAP). Preferably, the peroxyacid or salt thereofhas a melting point in the range of from 30° C. to 60° C.

The pre-formed peroxyacid or salt thereof can also be a peroxysulphonicacid or salt thereof, typically having a chemical structurecorresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁵ group can be linear or branched,substituted or unsubstituted; and Z is any suitable counter-ion thatachieves electric charge neutrality, preferably Z is selected fromhydrogen, sodium or potassium. Preferably R¹⁵ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably such bleachcomponents may be present in the compositions of the invention in anamount from 0.01 to 50% or from 0.1% to 20%.

(2) Sources of hydrogen peroxide include e.g., inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts such as those selected fromthe group consisting of sodium salts of perborate, percarbonate andmixtures thereof. When employed, inorganic perhydrate salts aretypically present in amounts of from 0.05 to 40 wt %, or 1 to 30 wt % ofthe overall composition and are typically incorporated into suchcompositions as a crystalline solid that may be coated. Suitablecoatings include: inorganic salts such as alkali metal silicate,carbonate or borate salts or mixtures thereof, or organic materials suchas water-soluble or dispersible polymers, waxes, oils or fatty soaps.Preferably such bleach components may be present in the compositions ofthe invention in an amount from 0.01 to 50%, or from 0.1% to 20%.

(3) Suitable bleach activators are those having R—(C═O)-L wherein R isan alkyl group, optionally branched, having, when the bleach activatoris hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12 carbon atomsand, when the bleach activator is hydrophilic, less than 6 carbon atomsor less than 4 carbon atoms; and L is leaving group. Examples ofsuitable leaving groups are benzoic acid and derivativesthereof—especially benzene sulphonate. Suitable bleach activatorsinclude dodecanoyl oxybenzene sulphonate, decanoyl oxybenzenesulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED) andnonanoyloxybenzene sulphonate (NOBS). Suitable bleach activators arealso disclosed in WO98/17767. While any suitable bleach activator may beemployed, in one aspect of the invention the subject cleaningcomposition may comprise NOBS, TAED or mixtures thereof. When present,the peracid and/or bleach activator is generally present in thecomposition in an amount of from 0.1 to 60 wt %, from 0.5 to 40 wt % orfrom 0.6 to 10 wt % based on the fabric and home care composition. Oneor more hydrophobic peracids or precursors thereof may be used incombination with one or more hydrophilic peracid or precursor thereof.Preferably such bleach components may be present in the compositions ofthe invention in an amount from 0.01% to 50%, or from 0.1% to 20%.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

(4) Diacyl peroxides—preferred diacyl peroxide bleaching species includethose selected from diacyl peroxides of the general formula:R¹—C(O)—OO—(O)C—R², in which R¹ represents a C₆-C₁₈ alkyl, preferablyC₆-C₁₂ alkyl group containing a linear chain of at least 5 carbon atomsand optionally containing one or more substituents (e.g.—N⁺(CH₃)_(3′)—COOH or —CN) and/or one or more interrupting moieties(e.g. —CONH— or —CH═CH—) interpolated between adjacent carbon atoms ofthe alkyl radical, and R² represents an aliphatic group compatible witha peroxide moiety, such that R¹ and R² together contain a total of 8 to30 carbon atoms. In one preferred aspect R¹ and R² are linearunsubstituted C₆-C₁₂ alkyl chains. Most preferably R¹ and R² areidentical. Diacyl peroxides, in which both R¹ and R² are C₆-C₁₂ alkylgroups, are particularly preferred. Preferably, at least one of, mostpreferably only one of, the R groups (R₁ or R₂), does not containbranching or pendant rings in the alpha position, or preferably neitherin the alpha nor beta positions or most preferably in none of the alphaor beta or gamma positions. In one further preferred embodiment the DAPmay be asymmetric, such that preferably the hydrolysis of R1 acyl groupis rapid to generate peracid, but the hydrolysis of R2 acyl group isslow.

The tetraacyl peroxide bleaching species is preferably selected fromtetraacyl peroxides of the general formula:R³—C(O)—OO—C(O)—(CH₂)n—C(O)—OO—C(O)—R³, in which R³ represents a C₁-C₉alkyl, or C₃-C₇, group and n represents an integer from 2 to 12, or 4 to10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species ispresent in an amount sufficient to provide at least 0.5 ppm, at least 10ppm, or at least 50 ppm by weight of the wash liquor. In a preferredembodiment, the bleaching species is present in an amount sufficient toprovide from 0.5 to 300 ppm, from 30 to 150 ppm by weight of the washliquor.

Preferably the bleach component comprises a bleach catalyst (5 and 6).

(5) Preferred are organic (non-metal) bleach catalysts include bleachcatalyst capable of accepting an oxygen atom from a peroxyacid and/orsalt thereof, and transferring the oxygen atom to an oxidizeablesubstrate. Suitable bleach catalysts include, but are not limited to:iminium cations and polyions; iminium zwitterions; modified amines;modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones andmixtures thereof.

Suitable iminium cations and polyions include, but are not limited to,N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared asdescribed in Tetrahedron (1992), 49(2), 423-38 (see e.g. compound 4, p.433); N-methyl-3,4-dihydroisoquinolinium p-toluene sulphonate, preparedas described in U.S. Pat. No. 5,360,569 (see e.g. Column 11, Example 1);and N-octyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in U.S. Pat. No. 5,360,568 (see e.g. Column 10, Example 3).

Suitable iminium zwitterions include, but are not limited to,N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared asdescribed in U.S. Pat. No. 5,576,282 (see e.g. Column 31, Example II);N-[2-(sulphooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, preparedas described in U.S. Pat. No. 5,817,614 (see e.g. Column 32, Example V);2-[3-[(2-ethylhexyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt, prepared as described in WO05/047264 (see e.g. page 18,Example 8), and2-[3-[(2-butyloctyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt.

Suitable modified amine oxygen transfer catalysts include, but are notlimited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can bemade according to the procedures described in Tetrahedron Letters(1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfercatalysts include, but are not limited to, sodium1-hydroxy-N-oxy-N-[2-(sulphooxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

Suitable N-sulphonyl imine oxygen transfer catalysts include, but arenot limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, preparedaccording to the procedure described in the Journal of Organic Chemistry(1990), 55(4), 1254-61.

Suitable N-phosphonyl imine oxygen transfer catalysts include, but arenot limited to,[R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)-phosphinicamide, which can be made according to the procedures described in theJournal of the Chemical Society, Chemical Communications (1994), (22),2569-70.

Suitable N-acyl imine oxygen transfer catalysts include, but are notlimited to, [N(E)]-N-(phenylmethylene)acetamide, which can be madeaccording to the procedures described in Polish Journal of Chemistry(2003), 77(5), 577-590.

Suitable thiadiazole dioxide oxygen transfer catalysts include but arenot limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, whichcan be made according to the procedures described in U.S. Pat. No.5,753,599 (Column 9, Example 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are notlimited to,(Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride,which can be made according to the procedures described in TetrahedronLetters (1994), 35(34), 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but arenot limited to,1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose asprepared in U.S. Pat. No. 6,649,085 (Column 12, Example 1).

Preferably, the bleach catalyst comprises an iminium and/or carbonylfunctional group and is typically capable of forming an oxaziridiniumand/or dioxirane functional group upon acceptance of an oxygen atom,especially upon acceptance of an oxygen atom from a peroxyacid and/orsalt thereof. Preferably, the bleach catalyst comprises an oxaziridiniumfunctional group and/or is capable of forming an oxaziridiniumfunctional group upon acceptance of an oxygen atom, especially uponacceptance of an oxygen atom from a peroxyacid and/or salt thereof.Preferably, the bleach catalyst comprises a cyclic iminium functionalgroup, preferably wherein the cyclic moiety has a ring size of from fiveto eight atoms (including the nitrogen atom), preferably six atoms.Preferably, the bleach catalyst comprises an aryliminium functionalgroup, preferably a bi-cyclic aryliminium functional group, preferably a3,4-dihydroisoquinolinium functional group. Typically, the iminefunctional group is a quaternary imine functional group and is typicallycapable of forming a quaternary oxaziridinium functional group uponacceptance of an oxygen atom, especially upon acceptance of an oxygenatom from a peroxyacid and/or salt thereof. In another aspect, thedetergent composition comprises a bleach component having a logP_(o/w)no greater than 0, no greater than −0.5, no greater than −1.0, nogreater than −1.5, no greater than −2.0, no greater than −2.5, nogreater than −3.0, or no greater than −3.5. The method for determininglogP_(o/w) is described in more detail below.

Typically, the bleach ingredient is capable of generating a bleachingspecies having a X_(SO) of from 0.01 to 0.30, from 0.05 to 0.25, or from0.10 to 0.20. The method for determining X_(SO) is described in moredetail below. For example, bleaching ingredients having anisoquinolinium structure are capable of generating a bleaching speciesthat has an oxaziridinium structure. In this example, the X_(SO) is thatof the oxaziridinium bleaching species.

Preferably, the bleach catalyst has a chemical structure correspondingto the following chemical formula:

wherein: n and m are independently from 0 to 4, preferably n and m areboth 0; each R¹ is independently selected from a substituted orunsubstituted radical selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fusedheterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto,carboxylic, and carboalkoxy radicals; and any two vicinal R¹substituents may combine to form a fused aryl, fused carbocyclic orfused heterocyclic ring; each R² is independently selected from asubstituted or unsubstituted radical independently selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl,aralkyl, alkylenes, heterocyclic ring, alkoxys, arylcarbonyl groups,carboxyalkyl groups and amide groups; any R² may be joined together withany other of R² to form part of a common ring; any geminal R² maycombine to form a carbonyl; and any two R² may combine to form asubstituted or unsubstituted fused unsaturated moiety; R³ is a C₁ to C₂₀substituted or unsubstituted alkyl; R⁴ is hydrogen or the moietyQ_(t)-A, wherein: Q is a branched or unbranched alkylene, t=0 or 1 and Ais an anionic group selected from the group consisting of OSO₃ ⁻, SO₃ ⁻,CO₂ ⁻, OCO₂ ⁻, OPO₃ ²⁻, OPO₃H⁻ and OPO₂ ⁻; R⁵ is hydrogen or the moiety—CR¹¹R¹²⁻Y-G_(b)-Y_(c)-[(CR⁹R¹⁰)_(y)—O]_(k)—R⁸, wherein: each Y isindependently selected from the group consisting of O, S, N—H, or N—R⁸;and each R⁸ is independently selected from the group consisting ofalkyl, aryl and heteroaryl, said moieties being substituted orunsubstituted, and whether substituted or unsubstituted said moietieshaving less than 21 carbons; each G is independently selected from thegroup consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁹ areindependently selected from the group consisting of H and C₁-C₄ alkyl;R¹¹ and R¹² are independently selected from the group consisting of Hand alkyl, or when taken together may join to form a carbonyl; b=0 or 1;c can=0 or 1, but c must=0 if b=0; y is an integer from 1 to 6; k is aninteger from 0 to 20; R⁶ is H, or an alkyl, aryl or heteroaryl moiety;said moieties being substituted or unsubstituted; and X, if present, isa suitable charge balancing counterion, preferably X is present when R⁴is hydrogen, suitable X, include but are not limited to: chloride,bromide, sulphate, methosulphate, sulphonate, p-toluenesulphonate,borontetraflouride and phosphate.

In one embodiment of the present invention, the bleach catalyst has astructure corresponding to general formula below:

wherein R¹³ is a branched alkyl group containing from three to 24 carbonatoms (including the branching carbon atoms) or a linear alkyl groupcontaining from one to 24 carbon atoms; preferably R¹³ is a branchedalkyl group containing from eight to 18 carbon atoms or linear alkylgroup containing from eight to eighteen carbon atoms; preferably R¹³ isselected from the group consisting of 2-propylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl;preferably R¹³ is selected from the group consisting of 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

Preferably the bleach component comprises a source of peracid inaddition to bleach catalyst, particularly organic bleach catalyst. Thesource of peracid may be selected from (a) pre-formed peracid; (b)percarbonate, perborate or persulfate salt (hydrogen peroxide source)preferably in combination with a bleach activator; and (c) perhydrolaseenzyme and an ester for forming peracid in situ in the presence of waterin a textile or hard surface treatment step.

When present, the peracid and/or bleach activator is generally presentin the composition in an amount of from 0.1 to 60 wt %, from 0.5 to 40wt % or from 0.6 to 10 wt % based on the composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

(6) Metal-containing Bleach Catalysts—The bleach component may beprovided by a catalytic metal complex. One type of metal-containingbleach catalyst is a catalyst system comprising a transition metalcation of defined bleach catalytic activity, such as copper, iron,titanium, ruthenium, tungsten, molybdenum, or manganese cations, anauxiliary metal cation having little or no bleach catalytic activity,such as zinc or aluminum cations, and a sequestrate having definedstability constants for the catalytic and auxiliary metal cations,particularly ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.Preferred catalysts are described in WO09/839,406, U.S. Pat. No.6,218,351 and WO00/012667. Particularly preferred are transition metalcatalyst or ligands therefore that are cross-bridged polydentate N-donorligands.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, e.g., the manganese-based catalysts disclosed inU.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described e.g.in U.S. Pat. No. 5,597,936; U.S. Pat. No. 5,595,967. Such cobaltcatalysts are readily prepared by known procedures, such as taught e.g.in U.S. Pat. No. 5,597,936 and U.S. Pat. No. 5,595,967.

Compositions herein may also suitably include a transition metal complexof ligands such as bispidones (U.S. Pat. No. 7,501,389) and/ormacropolycyclic rigid ligands—abbreviated as “MRLs”. As a practicalmatter, and not by way of limitation, the compositions and processesherein can be adjusted to provide on the order of at least one part perhundred million of the active MRL species in the aqueous washing medium,and will typically provide from 0.005 ppm to 25 ppm, from 0.05 ppm to 10ppm, or from 0.1 ppm to 5 ppm, of the MRL in the wash liquor.

Suitable transition-metals in the instant transition-metal bleachcatalyst include e.g. manganese, iron and chromium. Suitable MRLsinclude 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitabletransition metal MRLs are readily prepared by known procedures, such astaught e.g. in U.S. Pat. No. 6,225,464 and WO00/32601.

(7) Photobleaches—suitable photobleaches include e.g. sulfonated zincphthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes andmixtures thereof. Preferred bleach components for use in the presentcompositions of the invention comprise a hydrogen peroxide source,bleach activator and/or organic peroxyacid, optionally generated in situby the reaction of a hydrogen peroxide source and bleach activator, incombination with a bleach catalyst. Preferred bleach components comprisebleach catalysts, preferably organic bleach catalysts, as describedabove.

Particularly preferred bleach components are the bleach catalysts inparticular the organic bleach catalysts.

Fabric Hueing Agents—

The composition may comprise a fabric hueing agent. Suitable fabrichueing agents include dyes, dye-clay conjugates, and pigments. Suitabledyes include small molecule dyes and polymeric dyes. Suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of dyes falling into the Colour Index (C.I.) classificationsof Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, AcidViolet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof.

In another aspect, suitable small molecule dyes include small moleculedyes selected from the group consisting of Colour Index (Society ofDyers and Colourists, Bradford, UK) numbers Direct Violet 9, DirectViolet 35, Direct Violet 48, Direct Violet 51, Direct Violet 66, DirectViolet 99, Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue279, Acid Red 17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet15, Acid Violet 17, Acid Violet 24, Acid Violet 43, Acid Red 52, AcidViolet 49, Acid Violet 50, Acid Blue 15, Acid Blue 17, Acid Blue 25,Acid Blue 29, Acid Blue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80,Acid Blue 83, Acid Blue 90 and Acid Blue 113, Acid Black 1, Basic Violet1, Basic Violet 3, Basic Violet 4, Basic Violet 10, Basic Violet 35,Basic Blue 3, Basic Blue 16, Basic Blue 22, Basic Blue 47, Basic Blue66, Basic Blue 75, Basic Blue 159 and mixtures thereof. In anotheraspect, suitable small molecule dyes include small molecule dyesselected from the group consisting of Colour Index (Society of Dyers andColourists, Bradford, UK) numbers Acid Violet 17, Acid Violet 43, AcidRed 52, Acid Red 73, Acid Red 88, Acid Red 150, Acid Blue 25, Acid Blue29, Acid Blue 45, Acid Blue 113, Acid Black 1, Direct Blue 1, DirectBlue 71, Direct Violet 51 and mixtures thereof. In another aspect,suitable small molecule dyes include small molecule dyes selected fromthe group consisting of Colour Index (Society of Dyers and Colourists,Bradford, UK) numbers Acid Violet 17, Direct Blue 71, Direct Violet 51,Direct Blue 1, Acid Red 88, Acid Red 150, Acid Blue 29, Acid Blue 113 ormixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing conjugated chromogens (dye-polymerconjugates) and polymers with chromogens co-polymerized into thebackbone of the polymer and mixtures thereof.

In another aspect, suitable polymeric dyes include polymeric dyesselected from the group consisting of fabric-substantive colorants soldunder the name of Liquitint® (Milliken, Spartanburg, S.C., USA),dye-polymer conjugates formed from at least one reactive dye and apolymer selected from the group consisting of polymers comprising amoiety selected from the group consisting of a hydroxyl moiety, aprimary amine moiety, a secondary amine moiety, a thiol moiety andmixtures thereof. In still another aspect, suitable polymeric dyesinclude polymeric dyes selected from the group consisting of Liquitint®(Milliken, Spartanburg, S.C., USA) Violet CT, carboxymethyl cellulose(CMC) conjugated with a reactive blue, reactive violet or reactive reddye such as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme,Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product codeS-ACMC, alkoxylated triphenyl-methane polymeric colourants, alkoxylatedthiophene polymeric colourants, and mixtures thereof.

Preferred hueing dyes include the whitening agents found in WO08/87497.These whitening agents may be characterized by the following structure(I):

wherein R₁ and R₂ can independently be selected from:

a) [(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≦5; wherein y≧1; and wherein z=0 to 5;

b) R₁=alkyl, aryl or aryl alkyl and R₂=[(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≦10; wherein y≧1; and wherein z=0 to 5;

c) R₁=[CH₂CH₂(OR₃)CH₂OR₄] and R₂=[CH₂CH₂(OR₃)CH₂OR₄]

wherein R₃ is selected from the group consisting of H, (CH₂CH₂O)_(z)H,and mixtures thereof; and wherein z=0 to 10;

wherein R₄ is selected from the group consisting of (C₁-C₁₆)alkyl, arylgroups, and mixtures thereof; and

d) wherein R1 and R2 can independently be selected from the aminoaddition product of styrene oxide, glycidyl methyl ether, isobutylglycidyl ether, isopropylglycidyl ether, t-butyl glycidyl ether,2-ethylhexylgycidyl ether, and glycidylhexadecyl ether, followed by theaddition of from 1 to 10 alkylene oxide units.

A preferred whitening agent of the present invention may becharacterized by the following structure (II):

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≦5; wherein y≧1; and wherein z=0 to 5.

A further preferred whitening agent of the present invention may becharacterized by the following structure (III):

typically comprising a mixture having a total of 5 EO groups. Suitablepreferred molecules are those in Structure I having the followingpendant groups in “part a” above.

TABLE 2 R1 R2 R′ R″ x y R′ R″ x y a H H 3 1 H H 0 1 b H H 2 1 H H 1 1 c= b H H 1 1 H H 2 1 d = a H H 0 1 H H 3 1

Further whitening agents of use include those described in US2008/34511(Unilever). A preferred agent is “Violet 13”.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In another aspect, suitable dye clayconjugates include dye clay conjugates selected from the groupconsisting of one cationic/basic dye selected from the group consistingof C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I.Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through23, CI Basic Black 1 through 11, and a clay selected from the groupconsisting of Montmorillonite clay, Hectorite clay, Saponite clay andmixtures thereof. In still another aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of:Montmorillonite Basic Blue B7 C.I. 42595 conjugate, MontmorilloniteBasic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I.42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040 conjugate,Montmorillonite Basic Red R1 C.I. 45160 conjugate, Montmorillonite C.I.Basic Black 2 conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate,Hectorite Basic Blue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3C.I. 42555 conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate,Hectorite Basic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black2 conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof.

In another aspect, suitable pigments include pigments selected from thegroup consisting of Ultramarine Blue (C.I. Pigment Blue 29), UltramarineViolet (C.I. Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (anymixture of fabric hueing agents can be used). Suitable hueing agents aredescribed in more detail in U.S. Pat. No. 7,208,459. Preferred levels ofdye in compositions of the invention are 0.00001 to 0.5 wt %, or 0.0001to 0.25 wt %.

The concentration of dyes preferred in water for the treatment and/orcleaning step is from 1 ppb to 5 ppm, 10 ppb to 5 ppm or 20 ppb to 5ppm. In preferred compositions, the concentration of surfactant will befrom 0.2 to 3 g/l.

Encapsulates—

The composition may comprise an encapsulate. In one aspect, anencapsulate comprising a core, a shell having an inner and outersurface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a materialselected from the group consisting of perfumes; brighteners; dyes;insect repellants; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polyvinylalcohols, optionally containingother co-monomers; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamineformaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material anda shell, said shell at least partially surrounding said core material,is disclosed. At least 75%, 85% or 90% of said encapsulates may have afracture strength of from 0.2 MPa to 10 MPa, from 0.4 MPa to 5 MPa, from0.6 MPa to 3.5 MPa, or from 0.7 MPa to 3 MPa; and a benefit agentleakage of from 0% to 30%, from 0% to 20%, or from 0% to 5%.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle size from 1 microns to 80 microns, from 5 microns to 60microns, from 10 microns to 50 microns, or from 15 microns to 40microns.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle wall thickness from 30 nm to 250 nm, from 80 nm to 180 nm, orfrom 100 nm to 160 nm.

In one aspect, said encapsulates' core material may comprise a materialselected from the group consisting of a perfume raw material and/oroptionally a material selected from the group consisting of vegetableoil, including neat and/or blended vegetable oils including caster oil,coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil,palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconutoil, palm kernel oil, castor oil, lemon oil and mixtures thereof; estersof vegetable oils, esters, including dibutyl adipate, dibutyl phthalate,butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate,trioctyl phosphate and mixtures thereof; straight or branched chainhydrocarbons, including those straight or branched chain hydrocarbonshaving a boiling point of greater than about 80° C.; partiallyhydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, includingmonoisopropylbiphenyl, alkylated naphthalene, includingdipropylnaphthalene, petroleum spirits, including kerosene, mineral oiland mixtures thereof; aromatic solvents, including benzene, toluene andmixtures thereof; silicone oils; and mixtures thereof.

In one aspect, said encapsulates' wall material may comprise a suitableresin including the reaction product of an aldehyde and an amine,suitable aldehydes include, formaldehyde. Suitable amines includemelamine, urea, benzoguanamine, glycoluril, and mixtures thereof.Suitable melamines include methylol melamine, methylated methylolmelamine, imino melamine and mixtures thereof. Suitable ureas includedimethylol urea, methylated dimethylol urea, urea-resorcinol, andmixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with theencapsulates e.g. in a capsule slurry and/or added to a compositionbefore, during or after the encapsulates are added to such composition.Suitable capsules may be made by the following teaching ofUS2008/0305982; and/or US2009/0247449. Alternatively, suitable capsulesmay be purchased from Appleton Papers Inc. of Appleton, Wis. USA.

In a preferred aspect the composition can also comprise a depositionaid, preferably consisting of the group comprising cationic or nonionicpolymers. Suitable polymers include cationic starches, cationichydroxyethylcellulose, polyvinylformaldehyde, locust bean gum, mannans,xyloglucans, tamarind gum, polyethyleneterephthalate and polymerscontaining dimethylaminoethyl methacrylate, optionally with one ormonomers selected from the group comprising acrylic acid and acrylamide.

Perfumes—

In one aspect the composition comprises a perfume that comprises one ormore perfume raw materials selected from the group consisting of1,1′-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid, diethyl ester;(ethoxymethoxy)cyclododecane; 1,3-nonanediol, monoacetate;(3-methylbutoxy)acetic acid, 2-propenyl ester; beta-methylcyclododecaneethanol;2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol;oxacyclohexadecan-2-one; alpha-methyl-benzenemethanol acetate;trans-3-ethoxy-1,1,5-trimethylcyclohexane;4-(1,1-dimethylethyl)cyclohexanol acetate;dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; beta-methylbenzenepropanal; beta-methyl-3-(1-methylethyl)benzenepropanal;4-phenyl-2-butanone; 2-methylbutanoic acid, ethyl ester; benzaldehyde;2-methylbutanoic acid, 1-methylethyl ester;dihydro-5-pentyl-2(3H)furanone;(2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal;undecanal; 2-ethyl-alpha,alpha-dimethylbenzenepropanal; decanal;alpha,alpha-dimethylbenzeneethanol acetate; 2-(phenylmethylene)octanal;2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoicacid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one;2-pentylcyclopentanone; 3-oxo-2-pentyl cyclopentaneacetic acid, methylester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde;2-heptylcyclopentanone; 1-(4-methylphenyl)ethanone;(3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one;(3E)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one;benzeneethanol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde;10-undecenal; propanoic acid, phenylmethyl ester;beta-methylbenzenepentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene;alpha,alpha-dimethylbenzeneethanol;(2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid,phenylmethyl ester; cyclohexanepropanoic acid, 2-propenyl ester;hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene;1,5-dimethyl-bicyclo[3.2.1]octan-8-one oxime;4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde;3-buten-2-ol; 2-[[[2,4(or3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid, methylester; 8-cyclohexadecen-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol;2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-Octadien-1-ol;2-hydroxy-Benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenenitrile;4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one;tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; Acetic acid,(2-methylbutoxy)-, 2-propenyl ester; Benzoic acid, 2-hydroxy-,3-methylbutyl ester; 2-Buten-1-one,1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)—; Cyclopentanecarboxylicacid, 2-hexyl-3-oxo-, methyl ester; Benzenepropanal, 4-ethyl-.alpha.,.alpha.-dimethyl-; 3-Cyclohexene-1-carboxaldehyde,3-(4-hydroxy-4-methylpentyl)-; Ethanone,1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)-,[3R-(3.alpha., 3a.beta., 7.beta., 8a.alpha.)]-; Undecanal,2-methyl-2H-Pyran-2-one, 6-butyltetrahydro-; Benzenepropanal,4-(1,1-dimethylethyl)-.alpha.-methyl-; 2(3H)-Furanone, 5-heptyldihydro-;Benzoic acid, 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]-, methyl;Benzoic acid, 2-hydroxy-, phenylmethyl ester; Naphthalene, 2-methoxy-;2-Cyclopenten-1-one, 2-hexyl-; 2(3H)-Furanone, 5-hexyldihydro-;Oxiranecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester;2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; Benzenepentanol,.gamma.-methyl-; 3-Octanol, 3,7-dimethyl-;3,7-dimethyl-2,6-octadienenitrile; 3,7-dimethyl-6-octen-1-ol; Terpineolacetate; 2-methyl-6-methylene-7-Octen-2-ol, dihydro derivative;3a,4,5,6,7,7a-hexahydro-4,7-Methano-1H-inden-6-ol propanoate;3-methyl-2-buten-1-ol acetate; (Z)-3-Hexen-1-ol acetate;2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol;4-(octahydro-4,7-methano-5H-inden-5-ylidene)-butanal;3-2,4-dimethyl-cyclohexene-1-carboxaldehyde;1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-ethanone;2-hydroxy-benzoic acid, methyl ester; 2-hydroxy-benzoic acid, hexylester; 2-phenoxy-ethanol; 2-hydroxy-benzoic acid, pentyl ester;2,3-heptanedione; 2-hexen-1-ol; 6-Octen-2-ol, 2,6-dimethyl-; damascone(alpha, beta, gamma or delta or mixtures thereof),4,7-Methano-1H-inden-6-ol, 3a,4,5,6,7,7a-hexahydro-, acetate;9-Undecenal; 8-Undecenal; Isocyclocitral; Ethanone,1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-;3-Cyclohexene-1-carboxaldehyde, 3,5-dimethyl-;3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-Octadien-3-ol,3,7-dimethyl-; 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate; Lilial(p-t-Bucinal), and Cyclopentanone,2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and1-methyl-4-(1-methylethenyl)cyclohexene and mixtures thereof.

In one aspect the composition may comprise an encapsulated perfumeparticle comprising either a water-soluble hydroxylic compound ofmelamine-formaldehyde or modified polyvinyl alcohol. In one aspect theencapsulate comprises (a) an at least partially water-soluble solidmatrix comprising one or more water-soluble hydroxylic compounds,preferably starch; and (b) a perfume oil encapsulated by the solidmatrix.

In a further aspect the perfume may be pre-complexed with a polyamine,preferably a polyethylenimine so as to form a Schiff base.

Polymers—

The composition may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The composition may comprise one or more amphiphilic cleaning polymerssuch as the compound having the following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺-(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof.

The composition may comprise amphiphilic alkoxylated grease cleaningpolymers which have balanced hydrophilic and hydrophobic properties suchthat they remove grease particles from fabrics and surfaces. Specificembodiments of the amphiphilic alkoxylated grease cleaning polymers ofthe present invention comprise a core structure and a plurality ofalkoxylate groups attached to that core structure. These may comprisealkoxylated polyalkylenimines, preferably having an inner polyethyleneoxide block and an outer polypropylene oxide block.

Alkoxylated polycarboxylates such as those prepared from polyacrylatesare useful herein to provide additional grease removal performance. Suchmaterials are described in WO 91/08281 and PCT 90/01815. Chemically,these materials comprise polyacrylates having one ethoxy side-chain perevery 7-8 acrylate units. The side-chains are of the formula—(CH₂CH₂O)_(m) (CH₂)_(n)CH₃ wherein m is 2-3 and n is 6-12. Theside-chains are ester-linked to the polyacrylate “backbone” to provide a“comb” polymer type structure. The molecular weight can vary, but istypically in the range of about 2000 to about 50,000. Such alkoxylatedpolycarboxylates can comprise from about 0.05 wt % to about 10 wt % ofthe compositions herein.

The isoprenoid-derived surfactants of the present invention, and theirmixtures with other cosurfactants and other adjunct ingredients, areparticularly suited to be used with an amphilic graft co-polymer,preferably the amphilic graft co-polymer comprises (i) polyethyeleneglycol backbone; and (ii) and at least one pendant moiety selected frompolyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferredamphilic graft co-polymer is Sokalan HP22, supplied from BASF. Suitablepolymers include random graft copolymers, preferably a polyvinyl acetategrafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is preferably about 6000 andthe weight ratio of the polyethylene oxide to polyvinyl acetate is about40 to 60 and no more than 1 grafting point per 50 ethylene oxide units.

Carboxylate polymer—

The composition of the present invention may also include one or morecarboxylate polymers such as a maleate/acrylate random copolymer orpolyacrylate homopolymer. In one aspect, the carboxylate polymer is apolyacrylate homopolymer having a molecular weight of from 4,000 Da to9,000 Da, or from 6,000 Da to 9,000 Da.

Soil Release Polymer—

The composition of the present invention may also include one or moresoil release polymers having a structure as defined by one of thefollowing structures (I), (II) or (III):

(I) —[(OCHR¹—CHR²)_(a)—O—OC—Ar—OO-]_(d)

(II) —[(OCHR³—CHR⁴)_(b)—O—OC-sAr—CO-]_(e)

(III) —[(OCHR⁵—CHR⁶)_(c)—OR⁷]_(f)

wherein:

a, b and c are from 1 to 200;

d, e and f are from 1 to 50;

Ar is a 1,4-substituted phenylene;

sAr is 1,3-substituted phenylene substituted in position 5 with SO₃Me;

Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, mono-, di-, tri-, ortetraalkylammonium wherein the alkyl groups are C₁-C₁₈ alkyl or C₂-C₁₀hydroxyalkyl, or mixtures thereof;

R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H or C₁-C₁₈n-or iso-alkyl; and

R⁷ is a linear or branched C₁-C₁₈ alkyl, or a linear or branched C₂-C₃₀alkenyl, or a cycloalkyl group with 5 to 9 carbon atoms, or a C₈-C₃₀arylgroup, or a C₆-C₃₀arylalkyl group.

Suitable soil release polymers are polyester soil release polymers suchas Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6 suppliedby Rhodia. Other suitable soil release polymers include Texcarepolymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240,SRN300 and SRN325 supplied by Clariant. Other suitable soil releasepolymers are Marloquest polymers, such as Marloquest SL supplied bySasol.

Cellulosic Polymer—

The composition of the present invention may also include one or morecellulosic polymers including those selected from alkyl cellulose, alkylalkoxyalkyl cellulose, carboxyalkyl cellulose, alkyl carboxyalkylcellulose. In one aspect, the cellulosic polymers are selected from thegroup comprising carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixturesthereof. In one aspect, the carboxymethyl cellulose has a degree ofcarboxymethyl substitution from 0.5 to 0.9 and a molecular weight from100,000 Da to 300,000 Da.

Enzymes—

The composition may comprise one or more enzymes which provide cleaningperformance and/or fabric care benefits. Examples of suitable enzymesinclude, but are not limited to, hemicellulases, peroxidases, proteases,cellulases, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, mannanases, pectate lyases, keratinases, reductases,oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases,tannases, pentosanases, malanases, R-glucanases, arabinosidases,hyaluronidase, chondroitinase, laccase, and amylases, or mixturesthereof. A typical combination is an enzyme cocktail that may comprisee.g. a protease and lipase in conjunction with amylase. When present ina composition, the aforementioned additional enzymes may be present atlevels from 0.00001% to 2%, from 0.0001% to 1% or from 0.001% to 0.5%enzyme protein by weight of the composition.

In one aspect preferred enzymes would include a protease. Suitableproteases include metalloproteases and serine proteases, includingneutral or alkaline microbial serine proteases, such as subtilisins (EC3.4.21.62). Suitable proteases include those of animal, vegetable ormicrobial origin. In one aspect, such suitable protease may be ofmicrobial origin. The suitable proteases include chemically orgenetically modified mutants of the aforementioned suitable proteases.In one aspect, the suitable protease may be a serine protease, such asan alkaline microbial protease or/and a trypsin-type protease. Examplesof suitable neutral or alkaline proteases include:

(a) subtilisins (EC 3.4.21.62), including those derived from Bacillus,such as Bacillus lentus, B. alkalophilus, B. subtilis, B.amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described inU.S. Pat. No. 6,312,936; U.S. Pat. No. 5,679,630; U.S. Pat. No.4,760,025; U.S. Pat. No. 7,262,042 and WO09/021,867.

(b) trypsin-type or chymotrypsin-type proteases, such as trypsin (e.g.,of porcine or bovine origin), including the Fusarium protease describedin WO89/06270 and the chymotrypsin proteases derived from Cellumonasdescribed in WO05/052161 and WO05/052146.

(c) metalloproteases, including those derived from Bacillusamyloliquefaciens described in WO07/044,993.

Preferred proteases include those derived from Bacillus gibsonii orBacillus Lentus.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Savinase®, Primase®, Durazym®,Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®,Ovozyme®, Neutrase®, Everlase® and Esperase® by Novozymes A/S (Denmark),those sold under the tradename Maxatase®, Maxacal®, Maxapem®,Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®,Excellase® and Purafect OXP® by Genencor International, those sold underthe tradename Opticlean® and Optimase® by Solvay Enzymes, thoseavailable from Henkel/Kemira, namely BLAP (sequence shown in FIG. 29 ofU.S. Pat. No. 5,352,604 with the following mutationsS99D+S101R+S103A+V1041+G159S, hereinafter referred to as BLAP), BLAP R(BLAP with S3T+V41+V199M+V2051+L217D), BLAP X (BLAP with S3T+V41+V2051)and BLAP F49 (BLAP with S3T+V41+A194P+V199M+V2051+L217D)—all fromHenkel/Kemira; and KAP (Bacillus alkalophilus subtilisin with mutationsA230V+S256G+S259N) from Kao.

Suitable alpha-amylases include those of bacterial or fungal origin.Chemically or genetically modified mutants (variants) are included. Apreferred alkaline alpha-amylase is derived from a strain of Bacillus,such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillusstearothermophilus, Bacillus subtilis, or other Bacillus sp., such asBacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375 (U.S. Pat. No.7,153,818) DSM 12368, DSMZ no. 12649, KSM AP1378 (WO97/00324), KSM K36or KSM K38 (EP1022334). Preferred amylases include:

(a) the variants described in WO94/02597, WO94/18314, WO96/23874 andWO97/43424, especially the variants with substitutions in one or more ofthe following positions versus the enzyme listed as SEQ ID No. 2 inWO96/23874: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190,197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

(b) the variants described in U.S. Pat. No. 5,856,164, WO99/23211,WO96/23873, WO00/60060 and WO06/002643, especially the variants with oneor more substitutions in the following positions versus the AA560 enzymelisted as SEQ ID No. 12 in WO06/002643: 26, 30, 33, 82, 37, 106, 118,128, 133, 149, 150, 160, 178, 182, 186, 193, 203, 214, 231, 256, 257,258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314,315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445,446, 447, 450, 461, 471, 482, 484, preferably that also contain thedeletions of D183* and G184*.

(c) variants exhibiting at least 90% identity with SEQ ID No. 4 inWO06/002643, the wild-type enzyme from Bacillus SP722, especiallyvariants with deletions in the 183 and 184 positions and variantsdescribed in WO00/60060, which is incorporated herein by reference.

(d) variants exhibiting at least 95% identity with the wild-type enzymefrom Bacillus sp. 707 (SEQ ID NO:7 in U.S. Pat. No. 6,093,562),especially those comprising one or more of the following mutations M202,M208, S255, R172, and/or M261. Preferably said amylase comprises one ormore of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N and/orR172Q. Particularly preferred are those comprising the mutations M202Lor M202T.

(e) variants described in WO09/149,130, preferably those exhibiting atleast 90% identity with SEQ ID NO: 1 or SEQ ID NO:2 in WO09/149,130, thewild-type enzyme from Geobacillus Stearophermophilus or a truncatedversion thereof.

Suitable commercially available alpha-amylases include DURAMYL®,LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®,STAINZYME®, STAINZYME PLUS®, FUNGAMYL® and BAN®, (Novozymes A/S,Bagsvaerd, Denmark), KEMZYM® AT 9000 Biozym Biotech Trading GmbHWehlistrasse 27b A-1200 Wien Austria, RAPIDASE®, PURASTAR®, ENZYSIZE®,OPTISIZE HT PLUS®, POWERASE® and PURASTAR OXAM® (Genencor InternationalInc., Palo Alto, Calif.) and KAM® (Kao, 14-10 Nihonbashi Kayabacho,1-chome, Chuo-ku Tokyo 103-8210, Japan). In one aspect, suitableamylases include NATALASE®, STAINZYME® and STAINZYME PLUS® and mixturesthereof.

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065,455), cutinase from Magnaporthe grisea (WO10/107,560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084,412, WO13/033,318), Geobacillusstearothermophilus lipase (WO11/084,417), lipase from Bacillus subtilis(WO11/084,599), and lipase from Streptomyces griseus (WO11/150,157) andS. pristinaespiralis (WO12/137,147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109,500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111,143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100,028).

In one aspect, other preferred enzymes include microbial-derivedendoglucanases exhibiting endo-beta-1,4-glucanase activity(E.C.3.2.1.4), including a bacterial polypeptide endogenous to a memberof the genus Bacillus which has a sequence of at least 90%, 94%, 97% or99% identity to the amino acid sequence SEQ ID NO:2 in U.S. Pat. No.7,141,403 and mixtures thereof. Suitable endoglucanases are sold underthe tradenames Celluclean® and Whitezyme® (Novozymes A/S, Bagsvaerd,Denmark).

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway®, Xpect® and mannanases sold under the tradenamesMannaway® (all from Novozymes A/S, Bagsvaerd, Denmark), and Purabrite®(Genencor International Inc., Palo Alto, Calif.).

Dye Transfer Inhibiting Agents—

The compositions of the present invention may also include one or moredye transfer inhibiting agents. Suitable polymeric dye transferinhibiting agents include, but are not limited to, polyvinylpyrrolidonepolymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidoneand N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a composition, the dye transferinhibiting agents may be present at levels from 0.0001% to 10%, from0.01% to 5% or from 0.1% to 3% by weight of the composition.

Brighteners—

The compositions of the present invention can also contain additionalcomponents that may tint articles being cleaned, such as fluorescentbrighteners.

The composition may comprise C.I. fluorescent brightener 260 inalpha-crystalline form having the following structure:

In one aspect, the brightener is a cold water soluble brightener, suchas the C.I. fluorescent brightener 260 in alpha-crystalline form. In oneaspect the brightener is predominantly in alpha-crystalline form, whichmeans that typically at least 50 wt %, at least 75 wt %, at least 90 wt%, at least 99 wt %, or even substantially all, of the C.I. fluorescentbrightener 260 is in alpha-crystalline form.

The brightener is typically in micronized particulate form, having aweight average primary particle size of from 3 to 30 micrometers, from 3micrometers to 20 micrometers, or from 3 to 10 micrometers.

The composition may comprise C.I. fluorescent brightener 260 inbeta-crystalline form, and the weight ratio of: (i) C.I. fluorescentbrightener 260 in alpha-crystalline form, to (ii) C.I. fluorescentbrightener 260 in beta-crystalline form may be at least 0.1, or at least0.6. BE680847 relates to a process for making C.I. fluorescentbrightener 260 in alpha-crystalline form.

Commercial optical brighteners which may be useful in the presentinvention can be classified into subgroups, which include, but are notnecessarily limited to, derivatives of stilbene, pyrazoline, coumarin,carboxylic acid, methinecyanines, dibenzothiophene-5,5-dioxide, azoles,5- and 6-membered-ring heterocycles, and other miscellaneous agents.Examples of such brighteners are disclosed in “The Production andApplication of Fluorescent Brightening Agents”, M. Zahradnik, Publishedby John Wiley & Sons, New York (1982). Specific nonlimiting examples ofoptical brighteners which are useful in the present compositions arethose identified in U.S. Pat. No. 4,790,856 and U.S. Pat. No. 3,646,015.

A further suitable brightener has the structure below:

Suitable fluorescent brightener levels include lower levels of from0.01, from 0.05, from 0.1 or from 0.2 wt % to upper levels of 0.5 or0.75 wt %.

In one aspect the brightener may be loaded onto a clay to form aparticle. Silicate salts—The compositions of the present invention canalso contain silicate salts, such as sodium or potassium silicate. Thecomposition may comprise of from 0 wt % to less than 10 wt % silicatesalt, to 9 wt %, or to 8 wt %, or to 7 wt %, or to 6 wt %, or to 5 wt %,or to 4 wt %, or to 3 wt %, or even to 2 wt %, and from above 0 wt %, orfrom 0.5 wt %, or from 1 wt % silicate salt. A suitable silicate salt issodium silicate.

Dispersants—

The compositions of the present invention can also contain dispersants.Suitable water-soluble organic materials include the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

Enzyme Stabilizers—

Enzymes for use in compositions can be stabilized by various techniques.The enzymes employed herein can be stabilized by the presence ofwater-soluble sources of calcium and/or magnesium ions in the finishedfabric and home care compositions that provide such ions to the enzymes.In case of aqueous compositions comprising protease, a reversibleprotease inhibitor, such as a boron compound including borate, 4-formylphenylboronic acid, phenylboronic acid and derivatives thereof, orcompounds such as calcium formate, sodium formate and 1,2-propane diolcan be added to further improve stability.

Solvents—

Suitable solvents include water and other solvents such as lipophilicfluids. Examples of suitable lipophilic fluids include siloxanes, othersilicones, hydrocarbons, glycol ethers, glycerine derivatives such asglycerine ethers, perfluorinated amines, perfluorinated andhydrofluoroether solvents, low-volatility nonfluorinated organicsolvents, diol solvents, other environmentally-friendly solvents andmixtures thereof.

Structurant/Thickeners—

Structured liquids can either be internally structured, whereby thestructure is formed by primary ingredients (e.g. surfactant material)and/or externally structured by providing a three dimensional matrixstructure using secondary ingredients (e.g. polymers, clay and/orsilicate material). The composition may comprise a structurant, from0.01 wt % to 5 wt %, from 0.1 wt % to 2.0 wt % structurant. Thestructurant is typically selected from the group consisting ofdiglycerides and triglycerides, ethylene glycol distearate,microcrystalline cellulose, cellulose-based materials, microfibercellulose, hydrophobically modified alkali-swellable emulsions such asPolygel W30 (3VSigma), biopolymers, xanthan gum, gellan gum, andmixtures thereof. A suitable structurant includes hydrogenated castoroil, and non-ethoxylated derivatives thereof. A suitable structurant isdisclosed in U.S. Pat. No. 6,855,680. Such structurants have athread-like structuring system having a range of aspect ratios. Othersuitable structurants and the processes for making them are described inWO2010/034736.

Conditioning Agents—

The composition of the present invention may include a high meltingpoint fatty compound. The high melting point fatty compound usefulherein has a melting point of 25° C. or higher, and is selected from thegroup consisting of fatty alcohols, fatty acids, fatty alcoholderivatives, fatty acid derivatives, and mixtures thereof. Suchcompounds of low melting point are not intended to be included in thissection. Non-limiting examples of the high melting point compounds arefound in International Cosmetic Ingredient Dictionary, Fifth Edition,1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992.

The high melting point fatty compound is included in the composition ata level of from 0.1% to 40%, from 1% to 30%, from 1.5% to 16% by weightof the composition, from 1.5% to 8% in view of providing improvedconditioning benefits such as slippery feel during the application towet hair, softness and moisturized feel on dry hair.

The compositions of the present invention may contain a cationicpolymer. Concentrations of the cationic polymer in the compositiontypically range from 0.05% to 3%, in another embodiment from 0.075% to2.0%, and in yet another embodiment from 0.1% to 1.0%. Suitable cationicpolymers will have cationic charge densities of at least 0.5 meq/gm, inanother embodiment at least 0.9 meq/gm, in another embodiment at least1.2 meq/gm, in yet another embodiment at least 1.5 meq/gm, but in oneembodiment also less than 7 meq/gm, and in another embodiment less than5 meq/gm, at the pH of intended use of the composition, which pH willgenerally range from pH 3 to pH 9, in one embodiment between pH 4 and pH8. Herein, “cationic charge density” of a polymer refers to the ratio ofthe number of positive charges on the polymer to the molecular weight ofthe polymer. The average molecular weight of such suitable cationicpolymers will generally be between 10,000 and 10 million, in oneembodiment between 50,000 and 5 million, and in another embodimentbetween 100,000 and 3 million.

Suitable cationic polymers for use in the compositions of the presentinvention contain cationic nitrogen-containing moieties such asquaternary ammonium or cationic protonated amino moieties. Any anioniccounterions can be used in association with the cationic polymers solong as the polymers remain soluble in water, in the composition, or ina coacervate phase of the composition, and so long as the counterionsare physically and chemically compatible with the essential componentsof the composition or do not otherwise unduly impair compositionperformance, stability or aesthetics. Nonlimiting examples of suchcounterions include halides (e.g., chloride, fluoride, bromide, iodide),sulfate and methylsulfate.

Nonlimiting examples of such polymers are described in the CTFA CosmeticIngredient Dictionary, 3rd edition, edited by Estrin, Crosley, andHaynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc.,Washington, D.C. (1982)).

Other suitable cationic polymers for use in the composition includepolysaccharide polymers, cationic guar gum derivatives, quaternarynitrogen-containing cellulose ethers, synthetic polymers, copolymers ofetherified cellulose, guar and starch. When used, the cationic polymersherein are either soluble in the composition or are soluble in a complexcoacervate phase in the composition formed by the cationic polymer andthe anionic, amphoteric and/or zwitterionic surfactant componentdescribed hereinbefore. Complex coacervates of the cationic polymer canalso be formed with other charged materials in the composition. Suitablecationic polymers are described in U.S. Pat. No. 3,962,418; U.S. Pat.No. 3,958,581; and US2007/0207109.

The composition of the present invention may include a nonionic polymeras a conditioning agent. Polyalkylene glycols having a molecular weightof more than 1000 are useful herein. Useful are those having thefollowing general formula:

wherein R⁹⁵ is selected from the group consisting of H, methyl, andmixtures thereof. Conditioning agents, and in particular silicones, maybe included in the composition. The conditioning agents useful in thecompositions of the present invention typically comprise a waterinsoluble, water dispersible, non-volatile, liquid that formsemulsified, liquid particles. Suitable conditioning agents for use inthe composition are those conditioning agents characterized generally assilicones (e.g., silicone oils, cationic silicones, silicone gums, highrefractive silicones, and silicone resins), organic conditioning oils(e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinationsthereof, or those conditioning agents which otherwise form liquid,dispersed particles in the aqueous surfactant matrix herein. Suchconditioning agents should be physically and chemically compatible withthe essential components of the composition, and should not otherwiseunduly impair composition stability, aesthetics or performance.

The concentration of the conditioning agent in the composition should besufficient to provide the desired conditioning benefits. Suchconcentration can vary with the conditioning agent, the conditioningperformance desired, the average size of the conditioning agentparticles, the type and concentration of other components, and otherlike factors.

The concentration of the silicone conditioning agent typically rangesfrom 0.01% to 10%. Non-limiting examples of suitable siliconeconditioning agents, and optional suspending agents for the silicone,are described in U.S. Reissue Pat. No. 34,584; U.S. Pat. No. 5,104,646;U.S. Pat. No. 5,106,609; U.S. Pat. No. 4,152,416; U.S. Pat. No.2,826,551; U.S. Pat. No. 3,964,500; U.S. Pat. No. 4,364,837; U.S. Pat.No. 6,607,717; U.S. Pat. No. 6,482,969; U.S. Pat. No. 5,807,956; U.S.Pat. No. 5,981,681; U.S. Pat. No. 6,207,782; U.S. Pat. No. 7,465,439;U.S. Pat. No. 7,041,767; U.S. Pat. No. 7,217,777; US2007/0286837A1;US2005/0048549A1; US2007/0041929A1; GB849433; DE10036533, which are allincorporated herein by reference; Chemistry and Technology of Silicones,New York: Academic Press (1968); General Electric Silicone RubberProduct Data Sheets SE 30, SE 33, SE 54 and SE 76; Silicon Compounds,Petrarch Systems, Inc. (1984); and in Encyclopedia of Polymer Scienceand Engineering, vol. 15, 2d ed., pp 204-308, John Wiley & Sons, Inc.(1989).

The compositions of the present invention may also comprise from 0.05%to 3% of at least one organic conditioning oil as the conditioningagent, either alone or in combination with other conditioning agents,such as the silicones (described herein). Suitable conditioning oilsinclude hydrocarbon oils, polyolefins, and fatty esters. Also suitablefor use in the compositions herein are the conditioning agents describedby the Procter & Gamble Company in U.S. Pat. No. 5,674,478 and U.S. Pat.No. 5,750,122. Also suitable for use herein are those conditioningagents described in U.S. Pat. No. 4,529,586; U.S. Pat. No. 4,507,280;U.S. Pat. No. 4,663,158; U.S. Pat. No. 4,197,865; U.S. Pat. No.4,217,914; U.S. Pat. No. 4,381,919; and U.S. Pat. No. 4,422,853.

Hygiene and Malodour—

The compositions of the present invention may also comprise one or moreof zinc ricinoleate, thymol, quaternary ammonium salts such as Bardac®,polyethylenimines (such as Lupasol® from BASF) and zinc complexesthereof, silver and silver compounds, especially those designed toslowly release Ag⁺ or nano-silver dispersions.

Probiotics—

The compositions may comprise probiotics such as those described inWO2009/043709.

Suds Boosters—

If high sudsing is desired, suds boosters such as the C₁₀-C₁₆alkanolamides or C₁₀-C₁₄ alkyl sulphates can be incorporated into thecompositions, typically at 1%-10% levels. The C₁₀-C₁₄ monoethanol anddiethanol amides illustrate a typical class of such suds boosters. Useof such suds boosters with high sudsing adjunct surfactants such as theamine oxides, betaines and sultaines noted above is also advantageous.If desired, water-soluble magnesium and/or calcium salts such as MgCl₂,MgSO₄, CaCl₂, CaSO₄ and the like, can be added at levels of, typically,0.1%-2%, to provide additional suds and to enhance grease removalperformance.

Suds Suppressors—

Compounds for reducing or suppressing the formation of suds can beincorporated into the compositions of the present invention. Sudssuppression can be of particular importance in the so-called “highconcentration cleaning process” as described in U.S. Pat. No. 4,489,455and U.S. Pat. No. 4,489,574, and in front-loading-style washingmachines.

A wide variety of materials may be used as suds suppressors, and sudssuppressors are well known to those skilled in the art. See e.g. KirkOthmer Encyclopedia of Chemical Technology, Third Edition, Volume 7,pages 430-447 (John Wiley & Sons, Inc., 1979). Examples of sudssupressors include monocarboxylic fatty acid and soluble salts therein,high molecular weight hydrocarbons such as paraffin, fatty acid esters(e.g., fatty acid triglycerides), fatty acid esters of monovalentalcohols, aliphatic C₁₈-C₄₀ ketones (e.g., stearone), N-alkylated aminotriazines, waxy hydrocarbons preferably having a melting point belowabout 100° C., silicone suds suppressors, and secondary alcohols. Sudssupressors are described in U.S. Pat. No. 2,954,347; U.S. Pat. No.4,265,779; U.S. Pat. No. 4,265,779; U.S. Pat. No. 3,455,839; U.S. Pat.No. 3,933,672; U.S. Pat. No. 4,652,392; U.S. Pat. No. 4,978,471; U.S.Pat. No. 4,983,316; U.S. Pat. No. 5,288,431; U.S. Pat. No. 4,639,489;U.S. Pat. No. 4,749,740; U.S. Pat. No. 4,798,679; U.S. Pat. No.4,075,118; EP89307851.9; EP150872; and DOS 2,124,526.

For any detergent compositions to be used in automatic laundry washingmachines, suds should not form to the extent that they overflow thewashing machine. Suds suppressors, when utilized, are preferably presentin a “suds suppressing amount. By “suds suppressing amount” is meantthat the formulator of the composition can select an amount of this sudscontrolling agent that will sufficiently control the suds to result in alow-sudsing laundry detergent for use in automatic laundry washingmachines.

The compositions herein will generally comprise from 0% to 10% of sudssuppressor. When utilized as suds suppressors, monocarboxylic fattyacids, and salts therein, will be present typically in amounts up to 5%,by weight, of the detergent composition. Preferably, from 0.5% to 3% offatty monocarboxylate suds suppressor is utilized. Silicone sudssuppressors are typically utilized in amounts up to 2.0%, by weight, ofthe detergent composition, although higher amounts may be used.Monostearyl phosphate suds suppressors are generally utilized in amountsranging from 0.1% to 2%, by weight, of the composition. Hydrocarbon sudssuppressors are typically utilized in amounts ranging from 0.01% to5.0%, although higher levels can be used. The alcohol suds suppressorsare typically used at 0.2%-3% by weight of the finished compositions.

Water-Soluble Film—

The compositions of the present invention may also be encapsulatedwithin a water-soluble film. Preferred film materials are preferablypolymeric materials. The film material can e.g. be obtained by casting,blow-moulding, extrusion or blown extrusion of the polymeric material,as known in the art. Preferred polymers, copolymers or derivativesthereof suitable for use as pouch material are selected from polyvinylalcohols, polyvinyl pyrrolidone, polyalkylene oxides, acrylamide,acrylic acid, cellulose, cellulose ethers, cellulose esters, celluloseamides, polyvinyl acetates, polycarboxylic acids and salts,polyaminoacids or peptides, polyamides, polyacrylamide, copolymers ofmaleic/acrylic acids, polysaccharides including starch and gelatine,natural gums such as xanthum and carragum. More preferred polymers areselected from polyacrylates and water-soluble acrylate copolymers,methylcellulose, carboxymethylcellulose sodium, dextrin, ethylcellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, maltodextrin,polymethacrylates, and most preferably selected from polyvinyl alcohols,polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC),and combinations thereof. Preferably, the level of polymer in the pouchmaterial, e.g. a PVA polymer, is at least 60%. The polymer can have anyweight average molecular weight, preferably from about 1000 to1,000,000, more preferably from about 10,000 to 300,000 yet morepreferably from about 20,000 to 150,000. Mixtures of polymers can alsobe used as the pouch material.

Naturally, different film material and/or films of different thicknessmay be employed in making the compartments of the present invention. Abenefit in selecting different films is that the resulting compartmentsmay exhibit different solubility or release characteristics.

Preferred film materials are PVA films known under the MonoSol tradereference M8630, M8900, H8779 and those described in U.S. Pat. No.6,166,117 and U.S. Pat. No. 6,787,512 and PVA films of correspondingsolubility and deformability characteristics.

The film material herein can also comprise one or more additiveingredients. For example, it can be beneficial to add plasticisers, e.g.glycerol, ethylene glycol, diethyleneglycol, propylene glycol, sorbitoland mixtures thereof. Other additives include functional detergentadditives to be delivered to the wash water, e.g. organic polymericdispersants, etc.

Processes of Making the Compositions

The compositions of the present invention can be formulated into anysuitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in Applicants' examples andin U.S. Pat. No. 4,990,280; US20030087791A1; US20030087790A1;US20050003983A1; US20040048764A1; U.S. Pat. No. 4,762,636; U.S. Pat. No.6,291,412; US20050227891A1; EP1070115A2; U.S. Pat. No. 5,879,584; U.S.Pat. No. 5,691,297; U.S. Pat. No. 5,574,005; U.S. Pat. No. 5,569,645;U.S. Pat. No. 5,565,422; U.S. Pat. No. 5,516,448; U.S. Pat. No.5,489,392; U.S. Pat. No. 5,486,303 all of which are incorporated hereinby reference. The compositions of the invention or prepared according tothe invention comprise cleaning and/or treatment composition including,but not limited to, compositions for treating fabrics, hard surfaces andany other surfaces in the area of fabric and home care, including: aircare including air fresheners and scent delivery systems, car care,dishwashing, fabric conditioning (including softening and/orfreshening), laundry detergency, laundry and rinse additive and/or care,hard surface cleaning and/or treatment including floor and toilet bowlcleaners, granular or powder-form all-purpose or “heavy-duty” washingagents, especially cleaning detergents; liquid, gel or paste-formall-purpose washing agents, especially the so-called heavy-duty liquidtypes; liquid fine-fabric detergents; hand dishwashing agents or lightduty dishwashing agents, especially those of the high-foaming type;machine dishwashing agents, including the various tablet, granular,liquid and rinse-aid types for household and institutional use: car orcarpet shampoos, bathroom cleaners including toilet bowl cleaners; aswell as cleaning auxiliaries such as bleach additives and “stain-stick”or pre-treat types, substrate-laden compositions such as dryer addedsheets. Preferred are compositions and methods for cleaning and/ortreating textiles and/or hard surfaces, most preferably textiles. Thecompositions are preferably compositions used in a pre-treatment step ormain wash step of a washing process, most preferably for use in textilewashing step.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning compositions includingsoftening and/or freshening that may be in liquid, solid and/or dryersheet form; as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden compositions such asdryer added sheets. All of such compositions which are applicable may bein standard, concentrated or even highly concentrated form even to theextent that such compositions may in certain aspect be non-aqueous.

Method of Use

The present invention includes a method for cleaning and/or treating atextile or hard surface or other surface in fabric or home care. In apreferred aspect of the invention, the method comprises the step ofcontacting the surface to be treated in a pre-treatment step or mainwash step of a washing process, most preferably for use in a textilewashing step. In one embodiment of the invention the lipase variant andother components are added sequentially into the method for cleaningand/or treating the surface. Alternatively, the lipase variant and othercomponents are added simultaneously.

As used herein, washing includes but is not limited to, scrubbing, andmechanical agitation. Washing may be conducted with a foam compositionas described in WO08/101,958 and/or by applying alternating pressure(pressure/vacuum) as an addition or as an alternative to scrubbing andmechanical agitation. Drying of such surfaces or fabrics may beaccomplished by any one of the common means employed either in domesticor industrial settings. The cleaning compositions of the presentinvention are ideally suited for use in laundry applications.Accordingly, the present invention includes a method for laundering afabric. The method comprises the steps of contacting a fabric to belaundered with a said cleaning laundry solution comprising at least oneembodiment of Applicants' cleaning composition, cleaning additive ormixture thereof. The fabric may comprise most any fabric capable ofbeing laundered in normal consumer or institutional use conditions. Thesolution preferably has a pH of from 8 to 10.5. The compositions may beemployed at concentrations of from 500 ppm to 15,000 ppm in solution.The water temperatures typically range from 5° C. to 90° C. The water tofabric ratio is typically from 1:1 to 30:1.

In one aspect the invention relates to a method of using the variantproducing a cleaning composition. In on aspect the invention relates touse of the variant for cleaning.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising a polynucleotide of the presentinvention so as to express and produce the variant in recoverablequantities. The variant may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the variant may beused as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, and rheological properties,or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding a variant into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined,e.g., on the basis of when, where, and how the variant is desired to beexpressed. For instance, the expression of the gene encoding a variantmay be constitutive or inducible, or may be developmental, stage ortissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,e.g., described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,e.g., a promoter from storage sink tissues such as seeds, potato tubers,and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), orfrom metabolic sink tissues such as meristems (Ito et al., 1994, PlantMol. Biol. 24: 863-878), a seed specific promoter such as the glutelin,prolamin, globulin, or albumin promoter from rice (Wu et al., 1998,Plant Cell Physiol. 39: 885-889), a Vicia faba promoter from the leguminB4 and the unknown seed protein gene from Vicia faba (Conrad et al.,1998, J. Plant Physiol. 152: 708-711), a promoter from a seed oil bodyprotein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941), thestorage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a variant in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a variant. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. No. 6,395,966 and U.S. Pat. No. 7,151,204 (both of whichare herein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using,e.g., co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a variant can be introducedinto a particular plant variety by crossing, without the need for everdirectly transforming a plant of that given variety. Therefore, thepresent invention encompasses not only a plant directly regenerated fromcells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention. Crossingresults in the introduction of a transgene into a plant line by crosspollinating a starting line with a donor plant line. Non-limitingexamples of such steps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a variant ofthe present invention comprising: (a) cultivating a transgenic plant ora plant cell comprising a polynucleotide encoding the variant underconditions conducive for production of the variant; and (b) recoveringthe variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Specific Activity at Conditions with Low Calcium

Specific activity was measured for a reference lipase and lipasevariants at 25° C. temperature in 96 well microtiter plates (MTP) with 4ug/ml enzyme in final assay volume. The substrate was coated on thebottom of each well by adding 100 nmol olive oil (CAS no. 8001-25-0) and100 nmol pNP-decanoate (CAS no. 1956-09-8) solubilized in 99% hexane andevaporated for 2 hours in a fume-hood with the light switched off—thusmaking the assay plate. 20 ul of enzyme solution was transferred to 180ul buffer—50 mM Tris pH 8 with 1 mM CalCl₂ or 5 mM EDTA, on separate 96well MTP to make the enzyme dilution. 150 ul of each enzyme dilution wastransferred to the assay plate. Immediate placement in ELISA reader andabsorbance at 405 nm was followed for 15 minutes.

TABLE 3 shows the specific activity (micromol/mg/min) of the variants inthe presence of various concentrations of CaCl₂.

5 mM No Enzyme 1 mM CaCl₂ EDTA R Reference: T231R + N233R 42.6 2.4 1D62C + S83T + E87C + G91L + N92D + 39.5 13.8 F95Y + D96T + L97P + K98Q +T231R + N233R 2 S83T + E87T + G91L + N92D + F95Y + 36.4 9.8 D96T +L97P + K98Q + T231R + N233R 3 I86P + E87A + G91A + N94D + D96I + 21.64.1 L97F + K98D + T231R + N233R 4 S83T + R84G + I86D + E87Q + W89L +12.1 2.9 I90Q + G91L + N92E + L93T + N94S + F95Y + D96T + K98T + T231R +N233R

Example 2 Specific Activity at Various pH

Specific activity at various pH was investigated using essentially thesame conditions as described in example 1 except that the buffer was 100mM Tris +/−1 mM CalCl₂ or 5 mM EDTA and the absorbance was followed for10 minutes.

TABLE 4a shows the specific activity (micromol/mg/min) of the variantsin the presence of various concentrations of CaCl₂ at pH8.

1 mM CaCl₂ 5 mM EDTA No Specific activity deviation Specific activitydeviation R 26.86 0.09 3.47 0.12 1 10.43 3.02 4.69 1.25 2 25.43 0.186.09 2.43 3 13.34 0.17 3.95 0.48 4 1.87 0.00 1.10 0.25

TABLE 4b shows the specific activity (micromol/mg/min) of the variantsin the presence of various concentrations of CaCl₂ at pH9.

1 mM CaCl₂ 5 mM EDTA No Specific activity deviation Specific activitydeviation R 19.55 5.72 0.30 0.03 1 7.59 0.36 1.23 0.24 2 33.04 10.320.54 0.12 3 8.10 5.72 0.58 0.12 4 2.59 0.00 0.33 0.05

TABLE 4c shows the specific activity (micromol/mg/min) of the variantsin the presence of various concentrations of CaCl₂ at pH10.

1 mM CaCl₂ 5 mM EDTA No Specific activity deviation Specific activitydeviation R 26.59 10.88 0.08 0.01 1 5.07 0.04 0.46 0.02 2 17.11 3.760.24 0.04 3 19.66 2.69 0.12 0.12 4 0.80 0.26 0.04 0.05

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A lipase variant, comprising a substitution at one or more positionsin the lid helix corresponding to positions 186D,E,N,Q, E87C, 190D,E,Q,and N92D,E,Q of the mature polypeptide of SEQ ID NO: 2, wherein thevariant has lipase activity.
 2. The variant of claim 1, which is avariant of a parent lipase selected from the group consisting of: a. apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or 3; b. a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 1, or (ii) thefull-length complement of (i); c. a polypeptide encoded by apolynucleotide having at least 60% identity to the mature polypeptidecoding sequence of SEQ ID NO: 1; and d. a fragment of the maturepolypeptide of SEQ ID NO: 2 or 3, which has lipase activity.
 3. Thevariant of claim 1, which has at least 80% but less than 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 2 or
 3. 4. (canceled)5. The variant of claim 1, which comprises a substitution at a positioncorresponding to position 86 of the mature polypeptide of SEQ ID NO: 2.6. (canceled)
 7. The variant of claim 1, which comprises a substitutionat a position corresponding to position 87 of the mature polypeptide ofSEQ ID NO:
 2. 8. (canceled)
 9. The variant of claim 1, which comprises asubstitution at a position corresponding to position 90 of the maturepolypeptide of SEQ ID NO:
 2. 10. (canceled)
 11. The variant of claim 1,which comprises a substitution at a position corresponding to position92 of the mature polypeptide of SEQ ID NO:
 2. 12. (canceled)
 13. Thevariant of claim 1, which comprises a substitution at two positionscorresponding to any of positions 186D,E,N,Q, E87C, 190D,E,Q, andN92D,E,Q of the mature polypeptide of SEQ ID NO:
 2. 14. The variant ofclaim 1, which comprises a substitution at three positions correspondingto any of positions 186D,E,N,Q, E87C, 190D,E,Q, and N92D,E,Q of themature polypeptide of SEQ ID NO:
 2. 15. The variant of claim 1, whichcomprises a substitution at each position corresponding to positions186D,E,N,Q, E87C, 190D,E,Q, and N92D,E,Q of the mature polypeptide ofSEQ ID NO:
 2. 16. The variant of claim 1, which comprises one or moresubstitutions corresponding to positions selected from the groupconsisting of D62C; S83T; R84G; 186P; E87T,A,Q; W89L; G91L,A; L93T;N94D,S; F95Y; D96T,I; L97P,F; K98Q,D,T of the mature polypeptide of SEQID NO:
 2. 17. The variant of claim 1, which consists or comprises any ofthe sets of substitutions corresponding to positions in the maturepolypeptide of SEQ ID NO: 2: a)S83T+R84G+186D+E87T+W89L+190Q+G91L+N92D+L93T+F95Y+D96T+K98T; b)S83T+E87T+G91L+N92D+F95Y+D96T+L97P+K98Q; c)186P+E87A+G91A+N94D+D961+L97F+K98D; d)S83T+R84G+186D+E87A+W89L+190Q+G91L+N92E+L93T+N94S+F95Y+D96T+K98T; e)D62C+S83T+R84G+186D+E87C+W89L+190Q+G91L+N92D+L93T+F95Y+D96T+K98T; f)D62C+S83T+E87C+G91L+N92D+F95Y+D96T+L97P+K98Q; g)D62C+186P+E87C+G91A+N94D+D961+L97F+K98D; h)D62C+S83T+R84G+186D+E87C+W89L+190Q+G91L+N92E+L93T+N94S+F95Y+D96T+K98T;i)S83T+R84G+186D+E87T+W89L+190Q+G91L+N92D+L93T+F95Y+D96T+K98T+T231R+N233R;j) S83T+E87T+G91L+N92D+F95Y+D96T+L97P+K98Q+T231R+N233R; k)186P+E87A+G91A+N94D+D961+L97F+K98D+T231R+N233R; 1)S83T+R84G+186D+E87A+W89L+190Q+G91L+N92E+L93T+N94S+F95Y+D96T+K98T+T231R+N233R;m)D62C+S83T+R84G+186D+E87C+W89L+190Q+G91L+N92D+L93T+F95Y+D96T+K98T+T231R+N233R;n) D62C+S83T+E87C+G91L+N92D+F95Y+D96T+L97P+K98Q+T231R+N233R; o)D62C+186P+E87C+G91A+N94D+D961+L97F+K98D+T231R+N233R; or p)D62C+S83T+R84G+186D+E87C+W89L+190Q+G91L+N92E+L93T+N94S+F95Y+D96T+K98T+T231R+N233R.18. The variant of claim 1, which has an improved property relative tothe parent, wherein the improved property is selected from: improvedcatalytic rate; improved specific activity; improved substrate cleavage;improved opening of the lid; reduced calcium ion dependency; improvedbuilder tolerance, or improved wash performance.
 19. An isolatedpolynucleotide encoding the variant of claim
 1. 20. A nucleic acidconstruct comprising the polynucleotide of claim
 19. 21. An expressionvector comprising the polynucleotide of claim
 19. 22. A host cellcomprising the polynucleotide of claim
 19. 23. A method of producing alipase variant, comprising: a. cultivating the host cell of claim 22under conditions suitable for expression of the variant; and b.recovering the variant.
 24. (canceled)
 25. (canceled)
 26. A compositioncomprising the variant of claim
 1. 27. (canceled)
 28. A method ofcleaning a textile or hard surface or other surface in fabric and homecare comprising the steps of: a) contacting the surface with an aqueoussolution comprising (i) the variant of claim 1; b) rinsing and dryingthe textile or hard surface.
 29. (canceled)
 30. (canceled)